**2. Results and Discussion**

In the present study, we examined the preservation capacity of biopolymeric films with various incorporated plant-derived essential oils. Numerous studies have examined the benefits of using films and coatings based on biopolymers and essential oils; however, the long-term effect on their properties is not entirely known. Our results show that the storage of films considerably influences their properties. Although the films were kept under temperature- and humidity-controlled conditions, their mass varied after one year (Figure 1).

As depicted in Figure 1, the mass of all samples was reduced during the test period. Although samples were kept in silicone paper packaging, simulating a real storage environment, significant moisture loss had occurred after one year. The highest mass reduction was observed in sample 4 with 15% grapefruit EO (23.13%), and the lowest mass reduction was observed in sample 1 with 15% lemon EO (3.54%). The mass loss was associated with a significant reduction in film thickness (Figure 2).

**Figure 1.** Mass of biopolymeric films**. Figure 1.** Mass of biopolymeric films. ated with a significant reduction in film thickness (Figure 2).

4.00

5.00

6.00

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**Figure 2.** Sample thickness after one year of storage. T0, light brown bars; T1, blue bars. **Figure 2.** Sample thickness after one year of storage. T0, light brown bars; T1, blue bars.

**Figure 2.** Sample thickness after one year of storage. T0, light brown bars; T1, blue bars. The thickness of the films did not undergo major changes after one year compared to the initial period. The largest variation was observed in the films with the addition of The thickness of the films did not undergo major changes after one year compared to the initial period. The largest variation was observed in the films with the addition of cinnamon (from 94.67 to 76.67 µm and 99.67 to 71.00 µm, respectively) and cloves (from 95.67 µm to 83.33 µm (9) and from 66.67 µm to 53.67 µm for sample 10) oils. The thickness of the control sample was reduced by 9.7 µm. The determination and the results are of interest to developers who may want to produce such materials on a large scale.

The thickness of the films did not undergo major changes after one year compared to the initial period. The largest variation was observed in the films with the addition of The mass reduction in the films can be attributed to the elimination of water from the film matrix, which was also indicated by the reduced values of the water activity index. These values decreased by at least 50% for all samples (Table 1). A decrease in the water activity index can be beneficial in terms of preventing the development of microorganisms (which require at least a<sup>w</sup> > 0.7), but it can also alter the film microstructure, making it more brittle and fragile.


**Table 1.** Optical parameters and water activity index at the time of development (t0) and after one year of storage (t1).

\* The values represent the mean ± SD. Means that do not share the same superscript (a–p) are significantly different (*p* < 0.05).

In addition to dehydration, a reduction in sample density also occurred, totaling approximately one unit for samples 5, 6 and 14–18 with incorporated orange, chamomile, ginger and eucalyptus essential oils. Additionally, the density of the control sample (19) decreased by one unit (from 0.44 to 0.34 g/cm<sup>3</sup> ). The greatest reduction in density was observed in sample 4 (56.75%) composed of 15% grapefruit essential oil. Except for grapefruit (3, 4), mint (11, 12) and the control (19), all films exhibited increased opacity after one year of storage. These results were confirmed by a corresponding decrease in the transmittance parameter evaluated before and after storage (Figures 3 and 4).

As depicted in Figure 3, the transmittance values increased within 300–700 nm range. At 800 nm (visible light), films with 7.5% lemon (1), 15% cinnamon (8), 7.5% mint (11), 7.5 and 15% ginger (15,16), 15% eucalyptus (18) EOs and the control (19) exhibited increased transmittance, whereas in the remainder of the films, the transmission was decreased. This determination is important because it evaluates the ability of the material to resist UV radiation can degrade the incorporated product and even the foil, especially when it contains essential oil. The smallest variations were observed in the control sample (19) both before and after storage. After storage, the films were more resistant to the action of UV radiation, an effect strengthened by the increased opacity values after storage (Table 1).

The sample color was also influenced by storage. Except for samples with added mint oil (11, 12), in which the luminosity values increased during the storage period (from 90.61 and 91.9 to 92.25 and 92.18, respectively), for all other samples, the values were reduced but did not change significantly. The color deviation was more evident for samples 1–10 (∆E = 6.24–7.55) (Table 2). For samples 11–17, ∆E did not exceed 3.63. The smallest color deviation was observed in sample 12 with 15% mint oil (∆E = 0.244), and the largest color deviation was observed in sample 18 with 15% eucalyptus oil (∆E = 11.35).


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**Figure 3.** Transmittance values of samples (t0). **Figure 3.** Transmittance values of samples (t0).

**Figure 4.** The transmittance values of samples after one year of storage (t1). **Figure 4.** The transmittance values of samples after one year of storage (t1).

deviation was observed in sample 18 with 15% eucalyptus oil (ΔE = 11.35).

The sample color was also influenced by storage. Except for samples with added mint oil (11, 12), in which the luminosity values increased during the storage period (from 90.61 and 91.9 to 92.25 and 92.18, respectively), for all other samples, the values were reduced but did not change significantly. The color deviation was more evident for samples 1–10 (ΔE = 6.24–7.55) (Table 2). For samples 11–17, ΔE did not exceed 3.63. The smallest color deviation was observed in sample 12 with 15% mint oil (ΔE =0.244), and the largest color With some exceptions, the surface morphology of films with added oils was influenced by prolonged storage (Figure S1, Supplementary Files). The microstructure of films with 7.5 and 15% cinnamon (7,8), 7.5 and 15% clove (9,10) and 15% chamomile essential oils was negatively modified during storage. Changes also occurred in the structure of the control sample. The film with 15% grapefruit EO became rougher, indicating changes in the matrix, which was more homogeneous and compact prior to storage. The same pattern

**ΔE** 

1 92.73 ± 0.13 a,b 88.79 ± 0.18 b,c −5.51 ± 0.04 a −0.49 ± 0.02 a 11.42 ± 0.17 h 10.76 ± 0.31 f 6.42 ± 0.01<sup>f</sup> 2 92.23 ± 0.5 a,b,c 88.99 ± 0.16 b,c −5.82 ± 0.08 f,g −0.42 ± 0.24 a 13.43 ± 0.67 a,b,c 10.61 ± 0.30 f 6.53 ± 0.04<sup>f</sup> 3 92.12 ± 0.16 a,b,c 88.30 ± 0.40 b,c,d −5.90 ± 0.02 g,h −0.54 ± 0.02<sup>a</sup> 12.93 ± 0.33 c,d 11.33 ± 0.48 d,e,f 6.92 ± 0.01<sup>d</sup> 4 92.42 ± 0.28 a,b,c 88.87 ± 0.96 b,c −5.97 ± 0.08 h −0.61 ± 0.05<sup>a</sup> 14.08 ± 0.5 a 10.72 ± 0.53 f 7.55 ± 0.02<sup>b</sup> 5 92.45 ± 0.28 a,b,c 88.88 ± 0.73 b,c −5.78 ± 0.04 e,f −0.49 ± 0.04<sup>a</sup> 12.5 ± 0.19 d. e. f 8.85 ± 0.20 g 7.25 ± 0.05<sup>c</sup> 6 92.92 ± 0.31 a 87.17 ± 0.63 b −5.73 ± 0.02 d,e,f −0.48 ± 0.15<sup>a</sup> 12.82 ± 0.08 c,d,e 9.85 ± 0.73 f,g 6.97 ± 0.02<sup>d</sup> 7 92.47± 0.32 a,b,c 89.17 ± 0.30 b −5.68 ± 0.04 c,d,e −0.48 ± 0.01 a 11.36 ± 0.30 h 10.18 ± 0.08 d,e,f 6.51 ± 0.03<sup>f</sup> 8 92.57 ± 0.16 a,b,c 88.58 ± 0.92 b,c −5.66 ± 0.01 b,c,d −0.49 ± 0.02<sup>a</sup> 11.84 ± 0.30 f,g,h 10.95 ± 0.42 f 6.52 ± 0.03<sup>f</sup> 9 92.41 ± 0.39 a,b,c 88.50 ± 0.94 b,c,d −5.60 ± 0.01 b,c,d −0.47 ± 0.03 a 12.50 ± 0.27 d,e,f 11.14 ± 1.00 e,f 6.74 ± 0.02<sup>e</sup> 10 92.46 ± 0.21 a,b,c 88.31 ± 0.68 b,c,d −5.64 ± 0.03 b,c,d −0.52 ± 0.01 a 11.34 ± 0.31 h 11.22 ± 0.40 d,e,f 6.24 ± 0.06<sup>g</sup> 11 90.61 ± 0.64 d 92.25 ± 0.80 a −5.52 ± 0.03 a −5.78 ± 0.07<sup>c</sup> 13.30 ± 0.04 b,c 13.70,± 0.53 b,c 1.25 ± 0.01<sup>k</sup> 12 91.90 ± 0.48 b,c 92.18 ± 0.15 a −5.60 ± 0.03 a,b,c −5.79 ± 0.02<sup>c</sup> 12.50 ± 0.13 d,e,f 12.84 ± 0.52 c,d 0.24 ± 0.15<sup>m</sup> 13 92.65 ± 0.24 a,b 91.00 ± 0.64 a −5.59 ± 0.02 a,b,c −5.76 ± 0.01 c 12.51 ± 0.05 d,e,f 15.01 ± 0.40 c 3.63 ± 0.02<sup>h</sup> 14 92.34 ± 0.11 a,b,c 92.08 ± 0.28 a −5.67 ± 0.03 b,c,d −5.85 ± 0.03 c 12.18 ± 0.01 e,f,g 13.61 ± 0.37 b,c 0.85 ± 0.01<sup>l</sup> 15 92.54 ± 0.14 a,b,c 92.27 ± 0.24 a −5.68 ± 0.06 c,d,e −5.85 ± 0.03<sup>c</sup> 12.50 ± 0.38 d,e,f 13.17 ± 0.40 c 0.83 ± 0.02<sup>l</sup> 16 91.80 ± 0.40 c 91.83 ± 0.15 a −5.81 ± 0.01 f,g −5.76 ± 0.08<sup>c</sup> 13.82 ± 0.02 a,b 15.06 ± 0.27 b 1.61 ± 0.02<sup>j</sup> 17 92.41 ± 0.19 a,b,c 92.09 ± 0.44 a −5.68 ± 0.03 c,d,e −5.91 ± 0.10<sup>c</sup> 11.53 ± 0.15 g,h 12.84 ± 0.67 c,d,e 2.25 ± 0.04<sup>i</sup> 18 92.42 ± 0.33 a,b,c 86.97 ± 1.09 d −5.58 ± 0.04 a,b −5.18 ± 0.23<sup>b</sup> 11.75 ± 0.18 g,h 21.97 ± 0.8 a 11.35 ± 0.21<sup>a</sup>

**t0 t1 t0 t1 t0 t1**

**Table 2.** Evaluation of color parameters before (t0) and after storage (t1).

was observed for samples with 15% concentration of orange (6) and cinnamon (8) EOs. The film with 15% added clove oil (10) exhibited pores in its microstructure.

**Table 2.** Evaluation of color parameters before (t0) and after storage (t1).


L\*, lightness; a\*, green-to-red parameter; b\*, blue-to-yellow parameter. The values represent mean ± SD. Means that do not share the same superscript (a–m) are significantly different (*p* < 0.05).

Except for samples with added mint and ginger oil (11–14), all other films with essential oil presented a less homogeneous microstructure than the control sample. Thus, in order to obtain materials with superior physical properties, the matrix could be improved by increasing the amount of emulsifier in the film-forming solution (i.e., Tween 80).

The mechanical characteristics of the films were not influenced by the storage period. All samples subjected to testing showed a increased tensile strength after storage (Figure 5). The increase in breaking strength may be due to the amount of essential oil lost during storage in association with the overall decrease in the antioxidant activity of most films with added oil. The breaking strength of biopolymeric films has been shown to be affected by the addition of natural substances. For example, the mechanical performance was decreased with increasing content of natural substances added to films [31,32]. This effect was attributed to the plasticizing effect of EOs weakening the intermolecular interaction between polymer chains and increasing the ductility of the film [32,33].

Sample 14 (15% ginger) could not be tested to evaluate its mechanical properties as a result of the drying method used. Thus, a 15% concentration of ginger oil may require a increased amount of plasticizer or emulsifier in the composition.

As expected, the elongation at break of the films was negatively influenced during the storage period. The loss of water from the films unbalanced the matrix, so despite strengthened tear resistance, they showed considerably reduced elasticity (Figure 6).

The largest variation in elongation was observed in the lemon oil samples (16.7 (t0) to 1.51% (t1) and 18.28 (t0) to 1.91% (t1) for samples 1 and 2, respectively), regardless of the concentration. Large variations were also observed in the grapefruit, ginger and eucalyptus samples, with variations ranging between 37% and 50%. High variation was also observed in the control sample without the addition of essential oil. Therefore, the dehydration of the material did not depend on the nature of oil added but on the biopolymeric composition or environmental factors. However, the addition of orange, cinnamon, clove, mint and

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19 92.51 ± 0.12 a,b,c 87.47 ± 0.22 c,d −5.64 ± 0.02 b,c,d −0.62 ± 0.02<sup>a</sup> 11.32 ± 0.09 h 10.32 ± 0.56 f,g 7.14 ± 0.02<sup>c</sup>

L\*, lightness; a\*, green-to-red parameter; b\*, blue-to-yellow parameter. The values represent mean ± SD. Means that do not share the same superscript (a-m) are significantly different (*p* < 0.05).

The film with 15% added clove oil (10) exhibited pores in its microstructure.

between polymer chains and increasing the ductility of the film [32,33].

With some exceptions, the surface morphology of films with added oils was influenced by prolonged storage (Figure S1, Supplementary Files). The microstructure of films with 7.5 and 15% cinnamon (7,8), 7.5 and 15% clove (9,10) and 15% chamomile essential oils was negatively modified during storage. Changes also occurred in the structure of the control sample. The film with 15% grapefruit EO became rougher, indicating changes in the matrix, which was more homogeneous and compact prior to storage. The same pattern was observed for samples with 15% concentration of orange (6) and cinnamon (8) EOs.

Except for samples with added mint and ginger oil (11–14), all other films with essential oil presented a less homogeneous microstructure than the control sample. Thus, in order to obtain materials with superior physical properties, the matrix could be improved by increasing the amount of emulsifier in the film-forming solution (i.e., Tween 80).

The mechanical characteristics of the films were not influenced by the storage period. All samples subjected to testing showed a increased tensile strength after storage (Figure 5). The increase in breaking strength may be due to the amount of essential oil lost during storage in association with the overall decrease in the antioxidant activity of most films with added oil. The breaking strength of biopolymeric films has been shown to be affected by the addition of natural substances. For example, the mechanical performance was decreased with increasing content of natural substances added to films [31,32]. This effect was attributed to the plasticizing effect of EOs weakening the intermolecular interaction

chamomile essential oils maintained the elasticity of the samples, with minimal change relative to the initial period. Sample 14 (15% ginger) could not be tested to evaluate its mechanical properties as a result of the drying method used. Thus, a 15% concentration of ginger oil may require a increased amount of plasticizer or emulsifier in the composition.

**Figure 5.** Sample tensile strength**. Figure 5.** Sample tensile strength.

**Figure 6.** Sample elongation**. Figure 6.** Sample elongation.

activity from 8.36% to 17.27%.

The largest variation in elongation was observed in the lemon oil samples (16.7 (t0) to 1.51% (t1) and 18.28 (t0) to 1.91% (t1) for samples 1 and 2, respectively), regardless of the concentration. Large variations were also observed in the grapefruit, ginger and eucalyptus samples, with variations ranging between 37% and 50%. High variation was also observed in the control sample without the addition of essential oil. Therefore, the dehydration of the material did not depend on the nature of oil added but on the biopolymeric composition or environmental factors. However, the addition of orange, cinnamon, clove, mint and chamomile essential oils maintained the elasticity of the samples, with minimal change relative to the initial period. The antioxidant activity of most samples was significantly diminished after one year of storage (Figure 7). For example, the antioxidant activity of sample 2 with 15% lemon oil decreased from 26% to 1.9%, which was the most significant decrease among all samples The antioxidant activity of most samples was significantly diminished after one year of storage (Figure 7). For example, the antioxidant activity of sample 2 with 15% lemon oil decreased from 26% to 1.9%, which was the most significant decrease among all samples tested. A significant reduction in the antioxidant capacity was also observed in sample 8 with 15% cinnamon oil (from 23.36% to 2.28%). A modest decrease was also observed in sample 5 (7.5% orange oil) when the antioxidant activity dropped from 4.72% at *t*0 to 3.5% at *t*1. However, for a few samples, the antioxidant activity of the films increased with the addition of 15% orange (6), 7.5% cinnamon (7) and 7.5% clove (9) essential oils. As such, the incorporation of orange oil increased antioxidant activity from 11.09% to 17.73%, cinnamon oil increased antioxidant activity from 9.36% to 15.98% and clove oil increased said activity from 8.36% to 17.27%.

tested. A significant reduction in the antioxidant capacity was also observed in sample 8 with 15% cinnamon oil (from 23.36% to 2.28%). A modest decrease was also observed in sample 5 (7.5% orange oil) when the antioxidant activity dropped from 4.72% at *t0* to 3.5% at *t1*. However, for a few samples, the antioxidant activity of the films increased with the addition of 15% orange (6), 7.5% cinnamon (7) and 7.5% clove (9) essential oils. As such,

namon oil increased antioxidant activity from 9.36% to 15.98% and clove oil increased said

**Figure 7.** Graphical representation of DPPH radical scavenging activity of biopolymeric films before (red) and after storage (turquoise). **Figure 7.** Graphical representation of DPPH radical scavenging activity of biopolymeric films before (red) and after storage (turquoise).

The antioxidant effects of various essential oils on films are not entirely known, given the scarcity of studies examining their effects on biopolymeric matrices. However, the differential antioxidant effects of various natural oils have been well documented. For example, the antioxidant properties of clove and cinnamon essential oils are comparable to those of BHT (butylhydroxytoluene), a chemical substance known for its protective effect against oxidation. These results were attributed to the high eugenol and β-caryophyllene content of these oils [34], with clove essential oil being one of the most powerful natural antioxidants, with effects even superior to those of BHT or butylated hydroxyanisole [35]. The high antioxidant activity and radical scavenging effect of eugenol is the result of its phenolic hydroxyl groups, which remain stable during the film development process [36]. Similarly, linalool, another chemical compound present in orange and cinnamon oils (Table S1), has been recognized for its antioxidant and antibacterial activity [37,38]. When examining the antioxidant activity of 25 essential oils used for medicinal purposes, Wei and Shibamoto [39] showed that clove (52%) and cinnamon (23%) oils had antioxidant activity superior to that of chamomile (20%), anise (20%), rosemary (10%) and orange (9%) EOs. Moreover, according to their study, mint essential oil, along with sandalwood and bergamot, had exhibited pro-oxidant activity. Therefore, our results of increased antioxidant activity caused by clove, cinnamon and orange essential oils are somewhat in line with the general antioxidant activity effects of these oils, albeit in a different model and likely due to the high eugenol and linalool contents (Table S1, Supplementary Files), as well as the high antioxidant capacity of EOs. Our data cannot explain the concentration differences in the antioxidant effects of these oils, which may be attributed, in part, to their varied embedding properties into the a biopolymeric matrix; however, this would require further investigation. As shown in Table 3, microorganisms proliferated (*TC*) from t0 to t1 (samples 1–7, 9– The antioxidant effects of various essential oils on films are not entirely known, given the scarcity of studies examining their effects on biopolymeric matrices. However, the differential antioxidant effects of various natural oils have been well documented. For example, the antioxidant properties of clove and cinnamon essential oils are comparable to those of BHT (butylhydroxytoluene), a chemical substance known for its protective effect against oxidation. These results were attributed to the high eugenol and β-caryophyllene content of these oils [34], with clove essential oil being one of the most powerful natural antioxidants, with effects even superior to those of BHT or butylated hydroxyanisole [35]. The high antioxidant activity and radical scavenging effect of eugenol is the result of its phenolic hydroxyl groups, which remain stable during the film development process [36]. Similarly, linalool, another chemical compound present in orange and cinnamon oils (Table S1), has been recognized for its antioxidant and antibacterial activity [37,38]. When examining the antioxidant activity of 25 essential oils used for medicinal purposes, Wei and Shibamoto [39] showed that clove (52%) and cinnamon (23%) oils had antioxidant activity superior to that of chamomile (20%), anise (20%), rosemary (10%) and orange (9%) EOs. Moreover, according to their study, mint essential oil, along with sandalwood and bergamot, had exhibited pro-oxidant activity. Therefore, our results of increased antioxidant activity caused by clove, cinnamon and orange essential oils are somewhat in line with the general antioxidant activity effects of these oils, albeit in a different model and likely due to the high eugenol and linalool contents (Table S1, Supplementary Files), as well as the high antioxidant capacity of EOs. Our data cannot explain the concentration differences in the antioxidant effects of these oils, which may be attributed, in part, to their varied embedding properties into the a biopolymeric matrix; however, this would require further investigation.

14 and 19). Except for samples 15–18, which did not show a microbiological load before or after storage, the other films showed colony-forming units on their surfaces. Only samples with 7.5% essential oil of lemon, clove and mint showed contamination with coliforms bacteria, whereas samples 4 (15% grapefruit oil) and 13 (7.5% chamomile oil) were As shown in Table 3, microorganisms proliferated (*TC*) from t0 to t1 (samples 1–7, 9–14 and 19). Except for samples 15–18, which did not show a microbiological load before or after storage, the other films showed colony-forming units on their surfaces. Only samples with 7.5% essential oil of lemon, clove and mint showed contamination with coliforms bacteria, whereas samples 4 (15% grapefruit oil) and 13 (7.5% chamomile oil) were contaminated with *Staphylococcus aureus*. No *Escherichia coli*, enterococcus, *Listeria monocytogenes*, yeasts or molds developed on the surfaces of the biopolymeric materials.


**Table 3.** Microbiological assessments before (t0) and after storage (t1).

TC, total count; EC, Escherichia coli; ETC, Enterococcus; CF, coliform; YM, yeasts and molds; X-SA, Staphylococcus aureus; LM, Listeria monocytogenes. All results are expressed in CFU/g.
